PROJECT SUMMARY Excess weight gain contributes up to 65% of the risk of primary hypertension, and the increase in blood pressure (BP) in response to high-fat diet (HFD) is preceded by significant reductions in renal sodium (Na+) excretory capacity. Normal Na+ excretion is then only achieved at the expense of elevated renal perfusion pressure. Over time, the kidneys reset to require an elevated BP in order to continue to excrete a normal Na+ load. The knowledge of novel pathways regulating renal Na+ flux is therefore crucial to our success in optimizing therapy for major public health conditions such as obesity- hypertension. Recently, we reported that (1) direct renal infusion of ghrelin, the most potent, appetite-stimulating hormone in the body, activates GRs in the collecting duct (CD) to increase cAMP and ENaC-dependent Na+ reabsorption in rats; (2) renal infusion of a GR antagonist alone (to elucidate the actions of endogenous ghrelin), induces a robust natriuresis, indicating that physiological levels of circulating ghrelin regulate Na+ reabsorption under normal conditions; and (3) in HFD, renal GR expression is increased, and pharmacological blockade of intrarenal GRs prevents HFD- induced hypertension in rats. While these data establish some basic information regarding intrarenal GR expression and function, nothing is known about intrarenal production of ghrelin and its signaling pathways (Aim 1) or the contribution of GR-mediated antinatriuresis to the pathogenesis of HFD-induced hypertension (Aim 2). In this application, we propose studies in vitro using CD cells & in vivo using a HFD model of obesity- hypertension to study the mechanisms of the renal ghrelin-GR system. To isolate the role of the renal GR in chronic responses, we have developed a small inhibitory RNA (siRNA), that when infused chronically into the kidney, specifically transfects & knocks down CD GRs, while preserving GR expression in the other tissues. The major advantage of this over gene knockout is that it permits reduction in the gene expression without dangers of embryonic lethality and with less risk of compensatory gene regulation. We will also measure molecules released into the renal interstitium in response to renal manipulations of GR expression in conscious rats utilizing a novel microdialysis technique which permits analysis of mediators closer to target receptors and less prone to circulatory degradation. Thus, the ability to move fluidly between in vitro molecular and in vivo functional responses specific to the GR is a major significant aspect of the approach.